Primary adenosquamous carcinoma in a patient with Lynch syndrome

Adenosquamous colorectal carcinoma (ASCC) is rare and reported to represent between 0.06% and 0.20% of colorectal carcinoma (CRC).1Petrelli N.J. Valle A.A. Weber T.K. Rodriguez-Bigas M. Adenosquamous carcinoma of the colon and rectum.Dis Colon Rectum. 1996; 39: 1265-1268Crossref PubMed Scopus (55) Google Scholar Microsatellite instability-high (MSI-H) CRC caused by DNA mismatch repair (MMR) deficiency is detected in 15% of CRC, of which 12% are sporadic. These are caused by hypermethylation of MLH1 gene promoter, whilst the remaining 3% are inherited and associated with Lynch syndrome (LS).2Boland C.R. Goel A. Microsatellite instability in colorectal cancer.Gastroenterology. 2010; 138: 2073-2087Abstract Full Text Full Text PDF PubMed Scopus (1102) Google Scholar Recently, ASCC has been reported in association with MSI-H and LS, raising the possibility of MSI in the molecular pathogenesis and whether it may represent another histological subtype of MSI-H CRC. The molecular pathogenesis of ASCC remains poorly studied to date. Herein, we report the first case of ASCC with detailed molecular analysis by next-generation sequencing (NGS) of the glandular and squamous components, highlighting potential insights into the underlying molecular pathogenesis and a review of the literature. A 38-year-old previously fit and well female patient presented with sudden onset fever, nausea, vomiting and abdominal pain on a background of a 2-week history of diarrhoea. Computed tomography (CT) abdomen and pelvis scan revealed a heterogeneous 80×70 mm hepatic flexure lesion and she proceeded to have an extended right hemicolectomy with en bloc cholecystectomy, partial duodenectomy and pancreatectomy. Further history revealed that she belonged to a LS family with early-onset CRC in her mother and sister, and confirmed underlying MSH2 germline mutation. She had previously been offered but had declined genetic testing due to personal reasons. Post-operatively she was managed with adjuvant chemotherapy and during follow up of 9 months was negative for residual disease or recurrence. Histopathology examination showed a pT4bN0M1a, 95 mm, ulcerated hepatic flexure ASCC demonstrating areas of poorly-differentiated adenocarcinoma (Fig. 1A) and squamous cell carcinoma (Fig. 1B) with intimate admixture and zones of transition. There was direct invasion into the adherent duodenum and pancreas, no metastases in 22 regional lymph nodes, but metastatic tumour in one non-regional lymph node. The tumour was associated with a peritumoural Crohn's-like inflammatory reaction and tumour infiltrating lymphocytes (>4 per high power field). There was extensive lymphovascular invasion, including extramural venous invasion, and perineural invasion. Histochemical staining with Alcian blue/PASD showed apical, intraluminal and focal intracytoplasmic mucin within the glandular component and intermingled within squamous areas (Fig. 2A). Immunohistochemistry (IHC) showed the tumour to be positive for CK7 and negative for CK20 in both components. Variable CDX2 nuclear staining was observed in both components. The glandular component was positive for MOC31, BerEP4, CEA, EMA and MUC1 (Fig. 2B), whereas the squamous component stained for p40, CK5/6 and p63 (Fig. 2C), with cells in areas of transition demonstrating variable combined expression of both markers. The tumour showed preservation of MLH1 and PMS2 expression and loss of MSH2 and MSH6 DNA mismatch repair (MMR) protein expression. No BRAFV600E mutant protein was detected by IHC. MSI testing was performed using commercially available pentaplex MSI analysis kit (promega) containing five mononucleotide repeat markers (BAT-25, BAT-26, NR-21, NR-24 and MONO-27) and showed deletions in all five markers for both components, consistent with MSI-H tumour. NGS was performed using TruSight Tumour 26 gene kit (Illumina, USA) with pathogenic sequence variants detected in the KRAS, PIK3CA, TP53 and GNAS genes in both components (Table 1). Variants of clinical significance were not detected in target regions of other causally associated genes of PTEN, AKTI, SMAD4, MSH6, APC or FBXW7.Table 1Breakdown of the same pathogenic sequence variants detected in the glandular and squamous componentsGeneVAFRead depthHGVScHGVSpGlandular component KRAS18.174343c.38G>Ap.Gly13Asp PIK3CA17.914959c.3140A>Gp.His1047Arg GNAS17.874549c.2531G>Ap.Arg844His TP5318.073801c.541C>Tp.Arg181CysSquamous component KRAS11.4921c.38G>Ap.Gly13Asp PIK3CA12.87987c.3140A>Gp.His1047Arg GNAS10.031825c.2531G>Ap.Arg844His TP5311.142038c.541C>Tp.Arg181Cys Open table in a new tab ASCC was first reported in 1907 and is a rare subtype of colorectal carcinoma comprising of malignant glandular and squamous elements, either as separate areas within the tumour or admixed,3World health organization classification of tumours.in: Hamilton S.R. Aaltonen L.A. Pathology and Genetics of Tumours of the Digestive System. IARC Press, Lyon2000Google Scholar the squamous component consisting of more than just occasional small foci of squamous differentiation. Clinically, ASCC presents in a similar fashion to conventional colonic adenocarcinomas. Occasional patients with ASCC can present with paraneoplastic syndrome related hypercalcaemia.4Fujita T. Fukuda K. Nishi H. et al.Paraneoplastic hypercalcemia with adenosquamous carcinoma of the colon.Int J Clin Oncol. 2005; 10: 144-147Crossref PubMed Scopus (8) Google Scholar Most reported ASCC occur in the sixth to seventh decade and affect both males and females without ethnic predilection. Any area of the colon can be affected, although some studies report a predominance of proximal involvement.5Frizelle F.A. Hobday K.S. Batts K.P. Nelson H. Adenosquamous and squamous carcinoma of the colon and upper rectum: a clinical and histopathologic study.Dis Colon Rectum. 2001; 44: 341-346Crossref PubMed Scopus (100) Google Scholar Prior to a diagnosis of ASCC it is important to rule out other possible causes of malignant squamous elements in the large intestine, particularly metastasis or direct invasion, and confirm lack of continuity of the colorectal tumour with squamous lined sites or epithelia, including malignant transformation within squamous lined fistulous tract or invasion by anal SCC in distal sites (especially within 8 cm of the dentate line).6Dong Y. Wang J. Ma H. Zhou H. Lu G. Zhou X. Primary adenosquamous carcinoma of the colon: report of five cases.Surg Today. 2009; 39: 619-623Crossref PubMed Scopus (16) Google Scholar The pathogenesis of colorectal ASCC is poorly understood. Four hypotheses have been previously suggested including malignant transformation of embryological nests of ectodermal cells, squamous metaplasia, presence of pluripotent stem cells of endodermal origin capable of multidirectional differentiation and direct transformation/dedifferentiation of glandular epithelium.5Frizelle F.A. Hobday K.S. Batts K.P. Nelson H. Adenosquamous and squamous carcinoma of the colon and upper rectum: a clinical and histopathologic study.Dis Colon Rectum. 2001; 44: 341-346Crossref PubMed Scopus (100) Google Scholar It has also been proposed that an abnormal mucosal stimulus from conditions such as ulcerative colitis, schistosomiasis, radiation, or human papillomavirus (HPV) may allow squamous metaplasia and subsequent malignant transformation.1Petrelli N.J. Valle A.A. Weber T.K. Rodriguez-Bigas M. Adenosquamous carcinoma of the colon and rectum.Dis Colon Rectum. 1996; 39: 1265-1268Crossref PubMed Scopus (55) Google Scholar The role of HPV in ASCC is controversial with an association with high risk HPV genotype 16 reported by some investigators7Kong C.S. Welton M.L. Longacre T.A. Role of human papillomavirus in squamous cell metaplasia-dysplasia-carcinoma of the rectum.Am J Surg Pathol. 2007; 31: 919-925Crossref PubMed Scopus (39) Google Scholar but not others.6Dong Y. Wang J. Ma H. Zhou H. Lu G. Zhou X. Primary adenosquamous carcinoma of the colon: report of five cases.Surg Today. 2009; 39: 619-623Crossref PubMed Scopus (16) Google Scholar Traditionally, mixed tumours have been considered to potentially represent one of two subgroups: (1) composite tumours in which both components demonstrate intimate admixture or transition and suggestive of clonal relationship with divergent differentiation; or (2) collision tumours with coexistence and juxtaposition of two adjacent but histologically distinct tumours and suggestive of clonally unrelated tumours.8Duncan V.E. Harada S. Stevens T.M. Primary colon adenosquamous carcinoma in a patient with Lynch syndrome: a new histologic subtype associated with microsatellite instability?.Int J Surg Pathol. 2016; 24: 653-655Crossref PubMed Scopus (6) Google Scholar The former consideration has gained favour over time, and advances in molecular diagnostics have provided support for this notion, including adenosquamous carcinomas in extra-colonic sites. In our study, separate molecular analysis following microdissection on the glandular and squamous components of the tumour identified similar molecular abnormalities, supporting the concept of divergent differentiation from a common precursor rather than a collision tumour. The molecular abnormalities identified overlap with and include variants that have been commonly reported in conventional CRC. These findings raise the possibility of ASCC arising initially as adenocarcinoma prior to undergoing subsequent transdifferentiation, a concept that has been suggested in lung adenosquamous carcinomas.9Hou S. Zhou S. Qin Z. et al.Evidence, mechanism, and clinical relevance of the transdifferentiation from lung adenocarcinoma to squamous cell carcinoma.Am J Pathol. 2017; 187: 954-962Abstract Full Text Full Text PDF PubMed Scopus (32) Google Scholar The different histological features detected within the lesion almost certainly have unique molecular profiles and are driven by distinct sets of genetic and epigenetic alterations, as demonstrated for the different precursor colonic lesions such as sessile serrated adenomas and adenomatous polyps. We did not identify any additional underlying molecular genetic abnormalities within the squamous component that were not identified in the glandular component which might suggest possible underlying molecular genetic abnormalities that may be involved in driving phenotypic switch. However, the gene panel used is not sufficiently broad to provide further insight into this. We hope the information provided will serve as a starting point for future studies. Interestingly, a PIK3CA variant was identified, which has also been detected in adenosquamous carcinomas of other sites, including breast, lung and biliary tract, raising the possibility of PIK3CA inhibitors as novel treatment agents.10Bataillon G. Fuhrmann L. Girard E. et al.High rate of PIK3CA mutations but no TP53 mutations in low-grade adenosquamous carcinoma of the breast.Histopathology. 2018; 73: 273-283Crossref PubMed Scopus (16) Google Scholar Extensive literature review did not identify any studies reporting on the role of immunotherapy in ASCC. Recently, two cases of ASCC have been reported in association with MSI-H,8Duncan V.E. Harada S. Stevens T.M. Primary colon adenosquamous carcinoma in a patient with Lynch syndrome: a new histologic subtype associated with microsatellite instability?.Int J Surg Pathol. 2016; 24: 653-655Crossref PubMed Scopus (6) Google Scholar, 11Attiya A.A. Almaghraby H.Q. Satti M.B. An unusual variant of adenocarcinoma of the left colon associated with microsatellite instability: a case report.Int J Surg Pathol. 2017; 25: 550-554Google Scholar one of which was confirmed to represent a LS-related tumour. This raises interesting questions regarding the underlying molecular pathogenesis and its potential to represent another histological subtype associated with MSI-H and LS. Coincidentally, recent studies on the distribution of ASCC have noted a predilection for the proximal colon, a feature shared with MSI-H CRC. Furthermore, Petrelli et al.1Petrelli N.J. Valle A.A. Weber T.K. Rodriguez-Bigas M. Adenosquamous carcinoma of the colon and rectum.Dis Colon Rectum. 1996; 39: 1265-1268Crossref PubMed Scopus (55) Google Scholar reported that ASCC may be associated with increased risk of synchronous adenocarcinoma, a feature shared with LS-associated tumours. These findings add further circumstantial support for this notion. This case represents the third case of ASCC associated with MSI-H and the second confirmed case of LS-associated ASCC in peer reviewed literature. Although it is evident that at least a proportion of ASCC may be associated with MSI-H and LS, analogous to the situation in conventional CRC, the degree and strength of this relationship remains to be determined and the exact role of MSI-H in the molecular pathogenesis of ASCC. The role of MSI-H in the molecular pathogenesis of ASCC may have potential therapeutic implications if confirmed, raising the possibility of the consideration of immunotherapy in the treatment armamentarium.12Colle R. Cohen R. Cochereau D. et al.Immunotherapy and patients treated for cancer with microsatellite instability.Bull Cancer. 2017; 104: 42-51Crossref PubMed Scopus (45) Google Scholar As a corollary, documentation of efficacy of immunotherapy in adenosquamous carcinoma may provide indirect support of the potential of MSI-H in the pathogenesis of this rare tumour; however, to the best of our knowledge, there have been no definitive studies demonstrating efficacy of immunotherapy in adenosquamous carcinoma of the colon to date. ASCC is an aggressive subtype of colorectal carcinoma and is associated with worse prognosis when compared with usual colorectal carcinoma. This difference in stage-matched prognosis is less pronounced in early stage tumours without lymph node metastases but significantly worse with nodal metastasis.5Frizelle F.A. Hobday K.S. Batts K.P. Nelson H. Adenosquamous and squamous carcinoma of the colon and upper rectum: a clinical and histopathologic study.Dis Colon Rectum. 2001; 44: 341-346Crossref PubMed Scopus (100) Google Scholar The overall 5-year survival rate of ASCC is reported at 31% compared to 66% in adenocarcinomas, with the 5-year survival rate for node negative disease at 85%, falling to 23% in node positive disease.5Frizelle F.A. Hobday K.S. Batts K.P. Nelson H. Adenosquamous and squamous carcinoma of the colon and upper rectum: a clinical and histopathologic study.Dis Colon Rectum. 2001; 44: 341-346Crossref PubMed Scopus (100) Google Scholar Apart from well accepted poor prognostic factors such as metastasis, high stage and high grade disease, other suggested poor prognostic predictors include right sided lesions, ulcerated or annular carcinomas and association with ulcerative colitis.6Dong Y. Wang J. Ma H. Zhou H. Lu G. Zhou X. Primary adenosquamous carcinoma of the colon: report of five cases.Surg Today. 2009; 39: 619-623Crossref PubMed Scopus (16) Google Scholar Surgical resection is considered definitive treatment of ASCC. The role of adjuvant chemotherapy remains unclear. In conclusion, we report the first case of ASCC with detailed molecular analysis of the glandular and squamous components, and document only the second case of confirmed LS-related MSI-H ASCC. Our findings support the concept of divergent differentiation from a common precursor rather than a collision tumour, with a mutational profile that overlaps with and includes mutations identified in conventional CRC, raising the possibility of transdifferentiation. The exact role of MSI-H in ASCC and whether this could represent yet another histological subtype of CRC associated with MSI-H and LS requires further studies including molecular analysis, with an aim to identify potential triggers for the phenotypic switching. The authors state that there are no conflicts of interest to disclose.